WEEK ELEVEN & TWELVE (11/13/13) Cells were lysed by sonication, purified, and characterized. (11/8/13) Large culture was prepared, induced, re-suspended, and stored in Lysis Buffer. (11/7/13) Transformation results checked and a small culture started (11/6/13) Transforming new surrogate enzyme into BL21(DE3) cells
New Surrogate Clone Nanodrop
The Nanodrop concentration of the surrogate plasmid, E.coli DHFR in pNIC-Bsa4. The concentration on the tube was confirmed, about 250 ng/ul.
Results of Transformation From 11/7/13
Transformation results from 11/07/13. The surrogate plasmid, E. coli DHFR in pNIC-Bsa4, was transformed into BL21(DE3) cells and grown up overnight. These plates were then stored in the 4 degree celsius refrigerator.
Characterization Results Before Drying
Lane one: PageRule protein ladder. Lane two: flow-through step. Lane three: Wash step. Lane Four: Elution One. Lane Five: Elution Two, The gel is backwards, so lane one-lane five is from right to left. There is significant contamination in the form of a strong band, so FPLC will need to be undertaken.
WEEK NINE & TEN (10/23/13) Virtual screening- NADPH control dock and control library dock (10/23/13) Master plate and mini-prep (10/24/13) DNA sequencing (10/25/13) Virtual screening- NIH dock (10/26/13) Primary and secondary PCR (10/30/13) Secondary gel extraction (10/31/13) PCR squared off of secondary gel extract (11/1/13) pNIC-Bsa4 grown up
PCR squared off of secondary extract
PCR squared preformed off of secondary extract from 10/30/13. Lane one:1 kb NEB ladder. Lane two: PCR squared product. The brightest band is present at 1500 nt position, but there is some contamination.
Nanodrop Graph (trial one) from PCR squared sample after clean-up. The concentration and purity values are good.
Nanodrop Graph (trial two) from PCR squared sample after clean-up. The concentration and purity values are good.
Secondary gel extraction
A gel extraction was preformed on secondary PCR to then continue onto PCR squared. This was done in hopes of retaining high concentration values and purity.
Secondary PCR prior to gel extraction. Lane one:1 kb NEB ladder. Lane two-six:secondary PCR product that was successful from 10/26/13. The brightest bands are present at the 1500 nt position were excised.
Primary and Secondary PCR
Primary and secondary PCR was preformed again since the DNA sequencing results came back negative. This time, the protocol was altered in order to optimize the results.
Several secondary PCR samples each run under different conditions. Lane one:1 kb NEB ladder. Lane two:sample 1A. Lane three: sample 1B. Lane four: sample 2A. Lane five: sample 2B. Lane six: sample 3A. Lane seven: sample #1. Lane eight: sample #3. Lane nine: sample #4. Lane ten: sample #5. Successful samples (1A-3A, #3, and #4) were continued onto gel extraction.
Virtual screening-NIH dock
Since the results from the NADPH control dock were good, I decided to run a small library of ligands (~550).
DNA sequencing results
The results from DNA sequencing (sent 10/24/13) were not indicative of a positive clone. Again, only the beginning ~200 nt and the ending ~200 nt were cloned into pNIC-Bsa4. However, this phenomenon is random (sometimes only beginning clones in for example). We have not been able to conclude a reasonable explanation, but cloning will be attempted again.
Master Plate
A master plate was made off of the transformed cloning from 10/18/13 and mini-prepped to be sent to DNA sequencing.
Virtual Screening- NADPH and control library dock
Tony was able to obtain a crystallographic structure of L. major DHFR-TS from Dr. David Matthews (we came across references to this structure in literature about our target). A control dock on this new structure was performed and the results were promising (higher than our homology model).
Results from NADPH control dock.
Results from control library dock.
WEEK SEVEN & EIGHT
Nice work in the wet lab Renee. Good captions and analysis. Do you have any virtual data? Thank you. -Max 10/21/13
Overview (10/7/13) Transformation results from 10/5/13 were checked. Secondary PCR was made and ran on a gel. (10/8/13) Virtual Screening- control dock of NADPH. (10/9/13) Virtual Sreening- positive and negative control library dock. (10/8/13-10/9/13) Sending cloning results to DNA sequencing. (10/14/13) Two rounds of PCR squared and cleanup. (10/17/13) Gel Run of Clean PCR. Cut pNIC-Bsa4. (10/18/13) Cohesive End Generation and Transformation. (10/19/13) Transformation Results were checked.
Transformation Results from 10/18/13
DH5 alpha cells transformed with pNIC-Bsa4 (cloned with L. major DHFR-TS gene) grown on Agar+Kan+Suc plates and incubated for one day. Many, small colonies grew, which is a good indication.
Cohesive End Generation and Transformation
Protocol was carried out almost verbatim from the protocol except three transformation tubes were plated instead of two and the E. coli was heat-shocked for 45 seconds instead of 30 seconds. The three transformation tube concentrations were as follows:
Tube A: 2ul of vector and 4ul of insert
Tube B: 5ul of vector and 10 ul of insert
Tube C: 20 ul of vector and 17ul of insert
Gel Run of Clean PCR and PCR squared from 9/6/13
Lane one: 1kb NEB ladder. Lane two: Cleaned PCR squared from 10/14/13. Lane Three: Previously forgotten PCR squared from 9/6/13. Lane two showed multiple bands, which represents contamination. Lane three shows the brightest band at the correct nt length, 1500 nt. Therefore, the PCR product from 9/6/13 was cleaned up and used for cloning.
Cutting the pNIC-Bsa4
pNIC-Bsa4 from the previous two weeks was midiprepped, cut, and then cleaned up. The following are Nanodrop concentration from before and after cleanup. I modified the preparation protocol because 2.25 ng of my plasmid was about 40ul, which was over the final volume. I double the final concentration, and subsequently doubled the amount of reagents. Also, I let it cut for 3 hours.
Nanodrop concentration of pNIC-Bsa4 after midi-prep and before cutting/clean-up. This concentration is relatively good since some material is always lost in midi-prep.
Nanodrop concentration of pNIC-Bsa4 after cutting and after clean-up. This concentration is good (but not great) since only 20ng/ul was lost in cleanup.
Two Rounds of PCR squared and Cleanup
Two rounds of PCR squared were preformed off of secondary PCR from 10/7/13. One sample was cleaned and the other was run on a gel. However there were bands of contamination.
Lane one: 1kb NEB ladder. Lane two: PCR sqaured product. There is a band at 1500 nt (the correct position). However there was another bright band at the 2000 nt position.
DNA Sequencing Results
The results indicated that only parts of our gene cloned into the pNIC-Bsa4, the beginning 100-300 nt and the final 100-300 nt were clonning into our gene.
Virtual Screening-NADPH and Control Library Control Dock
For both control docks I used the 3inv homology model and superimposed 3inv to get the conformation of NADPH/the ligand and define the binding site. For the NADPH control dock this created a clash between NADPH and the homology model. However, this did not happen when I defined the binding site using NADPH and C50 (the ligand complexed into 3inv).
Results of NADPH dock:
GOLD scores of NADPH dock. Since this is a positive control dock, the score of 75 is considered low. This low score can be attributed to the clash.
Results of Control dock:
GOLD scores of control library. Some negative controls such as IDK scored higher than the natural substrate, DHF. Therefore, I will need to find a new way to define the active site.
Secondary PCR
Lane one: 1kb NEB ladder. Lane two: 15ul of secondary PCR sample. Brightest band is at 1500 nt position. However, there was another band at the 2000 nt position that was amplified during PCR squared.
Transformation Results from 10/7/13
There were three very large colonies. Each colony was mini-prepped, nano-dropped, and sent to sequencing.
WEEK FIVE & SIX
Good data but try to add captions and analysis. Thank you. -Max 10/07/2013
Overview (9/25/13) Two round of secondary PCR were run. (9/27/13) Positive and negative control ligands were found and 3D SDF files were concatenated. (10/2/13-10/4/13) New pNIC-Bsa4 was grown up to be used for cloning. (10/4/13) Four rounds of PCR squared were run and then extracted to use for cloning. pNIC-Bsa4 was also midi-prepped. (10/5/13) Cohesive End Generation and Transformation
Secondary PCR
Mine and Kevin E.'s secondary PCR (run on the same gel to save resources). First lane: 1kb NEB ladder. Second lane: primary PCR smear. Lane 3-4: secondary PCR product (one aliquot/lane from each tube). Lane 6-9: Kevin's results.
Positive and Negative Control Ligands Positive control ligands found via PDB structure similar to our target protein (ALL above 50% identity) and seeing what the protein was complexed with. The following compound identifiers were obtained as 3DSDF files from PubChem and then concatenated on the DDFE. 1) Natural Substrate= DHF 2) 1CX 3)UMP 4)C50 5)MTX 6)TMQ 7)2CY 8)DQ1 9)WRA 10)P128 11)P65 Negative control ligands (except for aspirin) were obtained by finding structures with similar physio chemical properties as the positive controls. All the compounds were obtained as 3DSDF files from PubChem and then concatenated on the DDFE.
Growing up more pNIC-Bsa4
DH5a E. coli cells transformed with pNIC-Bsa4 grown up on Agar and Kanamycin plates.
Four Rounds of PCR squared
One of four PCR squared gel results prior to extraction. Lanes 1-9: 37ul of PCR sqaured product. Lane 10: 1kb NEB ladder. All bands at the 1500 nt position were excised and then further extracted.
Nanodrop 1 result from Extracted PCR squared.
Nanodrop 2 result from Extracted PCR squared.
Concentration of pNIC-Bsa4 after midi-prep
WEEK THREE & FOUR
great work Renee - keep driving fowrard with the cloning - we need to get your clone soon so that you guys can move forward - Dr. B 100113
Overview (9/11/13) Research was resented in class. (9/13/13-9/14/13) New pNIC-Bsa4 was attempted to be grown up in a large-scale expression to be used for cloning. The attempt was unsuccessful probably due to the use of old colonies. (9/16/13-9/19/13) Homology models were made. (9/17/13) pNIC-Bsa4 grown during the summer was prepared for cloning. (9/18/13) A gel was run to ensure that the pNIC was successfully cut the previous day. (9/19/13) Cohesive end generation and transformation was done. Four rounds of PCR squared were preformed. (9/20/13) Transformation was unsuccessful. The four rounds of PCR squared were check on the gel, extracted, and then cleaned up due to low purity values. The resulting concentration was not very promising.
Homology Model
Homology model 3irm in orange superimposed over L. major DHFR-TS in yellow.
Homology model 3inv in green superimposed over L. major DHFR-TS in green.
pNIC-Bsa4 Used for Cloning
Recent Nanodrop data on pNIC-Bsa4 used for cloning since it had a relatively high concentration value and good purity values.
Virtual Gel
Virtual gel run of pNIC-Bsa4 cut with Bsa-I showing theoretical bands at 2,000 and 5,000 nt position. Obtained by running a custom digest on the NEB website.
Cut pNIC-Bsa4
Gel results of cut pNIC-Bsa4 used for cloning. Lane one is 1 kb ladder. Lane two is cut pNIC. Brightest bands are present at 2,000 and 5,00 nt position.
PCR Squared
Four rounds of PCR squared all run in full on 1% agarose gels with 1 kb ladder in lane one. Brightest bands on each gel are present at the 1500 nt position. The bands at the 1500 nt position were excised with a razor and then further extracted
PCR Squared Gel Extraction Nanodrop Results (Before Cleanup)
Nanodrop results from PCR squared gel extraction (1).
Nanodrop results from PCR squared gel extraction (2).
PCR Squared Clean Gel Extraction Nanodrop Results.
Nanodrop results from PCR squared gel extraction after cleanup (1).
Nanodrop results from PCR squared gel extraction after cleanup (2).
WEEK ONE & TWO Renee - good work. Dr. B 090913
Overview (9/2/13) New tail primers were ordered, a new oglio-mix was prepared. Primary and secondary PCR was preformed and tested on the gel. The results were a smear on primary PCR and a semi-bright band on the correct nuceotide position (1500nt). (9/6/13) PCR squared was preformed and tested on gel.Then gel extraction was performed on the PCR squared product since the band was at the correct position. The concentration after gel extraction was around 50ng/microliter. The 230nm. peak was high while the 260 nm. peak was low, which could account for poor cloning, but I have decided to continue to cloning next week.
Nanodrop Concentration after Gel Extraction
Nanodrop results of nucleotide concentration after gel extraction. (Second Trial)
Nanodrop results of nucleotide concentration after gel extraction. (First Trial)
Gel Extraction
PCR squared after gel extraction. The four bands at 1500 nt were excised and placed in the 2 ml snap-cap tubes for further extraction
PCR squared
PCR squared results. Lane One: NEB 1 kb ladder. Lanes Two-Five: 50 ul of PCR squared sample. The bands were at the correct position (1500 nt)
Primary and Secondary PCR
Primary and secondary PCR gel results. Lane One: NEB 1 kb ladder. Lane Two: Primary PCR smear (Tony). Lane Three: Secondary PCR (Tony). Lane Four: empty. Lane Five: Primary PCR smear. Lane Six: Secondary PCR. The bands of secondary PCR are in the correct position (1500 nt)
NEB 1 KB Ladder
1 kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are for 0.5 µg/lane.
WEEK FIVE
Primer Design (Putative acid phosphatase of H. pylori in pNIC-Bsa4) Forward Primer:
5’ TACTTCCAATCATGGTAAAAAAGACGCTGGCATC 3’ 34 bp GC Content 41.2%
0 mM Mg2+ Tm 62.1 oC 1.5 mM Mg2+ Tm 69.6 oC 2 mM Mg2+ Tm 70.1 oC 4 mM Mg2+ Tm 71.1oC 6 mM Mg2+ Tm 71.6 oC
Reverse Primer:
5’ AAAGCGTGGCAGAACAAAAAGTAACAGTAAAGGTGGATA 3’
Reverse complement it:
5’ TATCCACCTTTACTGTTACTTTTTGTTCTGCCACGCTT 3’ 38 bp GC Content 39.5% 0 mM Mg2+ Tm 63.7 oC 1.5 mM Mg2+ Tm 71.5 oC 2 mM Mg2+ Tm 71.9 oC 4 mM Mg2+ Tm 72.9 oC 6 mM Mg2+ Tm 73.3 oC
WEEK FOUR
Protein Purification: (PfDXR in pNic-BSSA4)
Nanodrop Results of elution one from protein purification ofPfDXR in pNic-BSSA4
Nanodrop Results of elution two from protein purification ofPfDXR in pNic-BSSA4
SDS-PAGE Gel Results:
Figure 1 – SDS-PAGE Gel Electrophoresis of His-Tag purified PfDXR in pNic-BSSA4; Well 1: Thermo Scientific Prestained Protein Ladder 10-170kDA; Well 2: Cell lysate before Induction; Well 3: Cell lysate after induction; Well 4: Supernatant of Lysed Cells; Well 5: Flowthrough of supernatant flushed with Ni-NTA resin/buffer mix; Well 6: Wash fraction with 20 mM Imidazole; Well 7: Elution 1, using 250mM Imidazole; Well 8: Elution 2, using 250mM Imidazole
WEEK THREE
Third PCR: (pmCHERRY)
Agarose gel run of third PCR; Wells 1 & 6: ladder, Wells 2-5: pmCHERRY (Samples 1, 2, 3, 4 respectively), Wells 7-10: pmCHERRY (Samples 5, 6, 7, 8 respectively)
Large-Scale Protein Expression: (PfDXR in pNic-BSSA4) pellet weight=2.8 g.
Second PCR: (pgbr22)
Agarose gel run of second PCR; Wells 1 & 6: ladder, Wells 2-5: pgbr22 (Samples A, B, C, D respectively), Wells 7-10: pgbr22 (Samples A, B, C, D respectively)
_
WEEK TWO
First PCR Results:
Agarose gel run of first PCR; Wells 1 & 6: ladder, Wells 2-5: pmCHERRY (Samples A, B, C, D respectively), Wells 7-10: pNIC (Samples A, B, C, D respectively)
Midi-Prep DNA Sequence: (92015) NNNNNNNNNNNNNNNTNNACTTTAGANGAGATNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATNTGGGT
FALL 2013
WEEK ELEVEN & TWELVE
(11/13/13) Cells were lysed by sonication, purified, and characterized.
(11/8/13) Large culture was prepared, induced, re-suspended, and stored in Lysis Buffer.
(11/7/13) Transformation results checked and a small culture started
(11/6/13) Transforming new surrogate enzyme into BL21(DE3) cells
New Surrogate Clone Nanodrop
Results of Transformation From 11/7/13
Characterization Results Before Drying
WEEK NINE & TEN
(10/23/13) Virtual screening- NADPH control dock and control library dock
(10/23/13) Master plate and mini-prep
(10/24/13) DNA sequencing
(10/25/13) Virtual screening- NIH dock
(10/26/13) Primary and secondary PCR
(10/30/13) Secondary gel extraction
(10/31/13) PCR squared off of secondary gel extract
(11/1/13) pNIC-Bsa4 grown up
PCR squared off of secondary extract
Secondary gel extraction
A gel extraction was preformed on secondary PCR to then continue onto PCR squared. This was done in hopes of retaining high concentration values and purity.
Primary and Secondary PCR
Primary and secondary PCR was preformed again since the DNA sequencing results came back negative. This time, the protocol was altered in order to optimize the results.
Virtual screening-NIH dock
Since the results from the NADPH control dock were good, I decided to run a small library of ligands (~550).
DNA sequencing results
The results from DNA sequencing (sent 10/24/13) were not indicative of a positive clone. Again, only the beginning ~200 nt and the ending ~200 nt were cloned into pNIC-Bsa4. However, this phenomenon is random (sometimes only beginning clones in for example). We have not been able to conclude a reasonable explanation, but cloning will be attempted again.
Master Plate
A master plate was made off of the transformed cloning from 10/18/13 and mini-prepped to be sent to DNA sequencing.
Virtual Screening- NADPH and control library dock
Tony was able to obtain a crystallographic structure of L. major DHFR-TS from Dr. David Matthews (we came across references to this structure in literature about our target). A control dock on this new structure was performed and the results were promising (higher than our homology model).
WEEK SEVEN & EIGHT
Nice work in the wet lab Renee. Good captions and analysis. Do you have any virtual data? Thank you. -Max 10/21/13
Overview
(10/7/13) Transformation results from 10/5/13 were checked. Secondary PCR was made and ran on a gel.
(10/8/13) Virtual Screening- control dock of NADPH.
(10/9/13) Virtual Sreening- positive and negative control library dock.
(10/8/13-10/9/13) Sending cloning results to DNA sequencing.
(10/14/13) Two rounds of PCR squared and cleanup.
(10/17/13) Gel Run of Clean PCR. Cut pNIC-Bsa4.
(10/18/13) Cohesive End Generation and Transformation.
(10/19/13) Transformation Results were checked.
Transformation Results from 10/18/13
Cohesive End Generation and Transformation
Protocol was carried out almost verbatim from the protocol except three transformation tubes were plated instead of two and the E. coli was heat-shocked for 45 seconds instead of 30 seconds. The three transformation tube concentrations were as follows:
Tube A: 2ul of vector and 4ul of insert
Tube B: 5ul of vector and 10 ul of insert
Tube C: 20 ul of vector and 17ul of insert
Gel Run of Clean PCR and PCR squared from 9/6/13
Cutting the pNIC-Bsa4
pNIC-Bsa4 from the previous two weeks was midiprepped, cut, and then cleaned up. The following are Nanodrop concentration from before and after cleanup. I modified the preparation protocol because 2.25 ng of my plasmid was about 40ul, which was over the final volume. I double the final concentration, and subsequently doubled the amount of reagents. Also, I let it cut for 3 hours.
Two Rounds of PCR squared and Cleanup
Two rounds of PCR squared were preformed off of secondary PCR from 10/7/13. One sample was cleaned and the other was run on a gel. However there were bands of contamination.
DNA Sequencing Results
The results indicated that only parts of our gene cloned into the pNIC-Bsa4, the beginning 100-300 nt and the final 100-300 nt were clonning into our gene.
Virtual Screening-NADPH and Control Library Control Dock
For both control docks I used the 3inv homology model and superimposed 3inv to get the conformation of NADPH/the ligand and define the binding site. For the NADPH control dock this created a clash between NADPH and the homology model. However, this did not happen when I defined the binding site using NADPH and C50 (the ligand complexed into 3inv).
Results of NADPH dock:
Results of Control dock:
Secondary PCR
Transformation Results from 10/7/13
There were three very large colonies. Each colony was mini-prepped, nano-dropped, and sent to sequencing.
WEEK FIVE & SIX
- Good data but try to add captions and analysis. Thank you. -Max 10/07/2013
Overview(9/25/13) Two round of secondary PCR were run.
(9/27/13) Positive and negative control ligands were found and 3D SDF files were concatenated.
(10/2/13-10/4/13) New pNIC-Bsa4 was grown up to be used for cloning.
(10/4/13) Four rounds of PCR squared were run and then extracted to use for cloning. pNIC-Bsa4 was also midi-prepped.
(10/5/13) Cohesive End Generation and Transformation
Secondary PCR
Positive and Negative Control Ligands
Positive control ligands found via PDB structure similar to our target protein (ALL above 50% identity) and seeing what the protein was complexed with. The following compound identifiers were obtained as 3DSDF files from PubChem and then concatenated on the DDFE.
1) Natural Substrate= DHF
2) 1CX
3)UMP
4)C50
5)MTX
6)TMQ
7)2CY
8)DQ1
9)WRA
10)P128
11)P65
Negative control ligands (except for aspirin) were obtained by finding structures with similar physio chemical properties as the positive controls. All the compounds were obtained as 3DSDF files from PubChem and then concatenated on the DDFE.
Growing up more pNIC-Bsa4
Four Rounds of PCR squared
Concentration of pNIC-Bsa4 after midi-prep
WEEK THREE & FOUR
great work Renee - keep driving fowrard with the cloning - we need to get your clone soon so that you guys can move forward - Dr. B 100113
Overview
(9/11/13) Research was resented in class.
(9/13/13-9/14/13) New pNIC-Bsa4 was attempted to be grown up in a large-scale expression to be used for cloning. The attempt was unsuccessful probably due to the use of old colonies.
(9/16/13-9/19/13) Homology models were made.
(9/17/13) pNIC-Bsa4 grown during the summer was prepared for cloning.
(9/18/13) A gel was run to ensure that the pNIC was successfully cut the previous day.
(9/19/13) Cohesive end generation and transformation was done. Four rounds of PCR squared were preformed.
(9/20/13) Transformation was unsuccessful. The four rounds of PCR squared were check on the gel, extracted, and then cleaned up due to low purity values. The resulting concentration was not very promising.
Homology Model
pNIC-Bsa4 Used for Cloning
Virtual Gel
Cut pNIC-Bsa4
PCR Squared
PCR Squared Gel Extraction Nanodrop Results (Before Cleanup)
PCR Squared Clean Gel Extraction Nanodrop Results.
WEEK ONE & TWO
Renee - good work. Dr. B 090913
Overview
(9/2/13) New tail primers were ordered, a new oglio-mix was prepared. Primary and secondary PCR was preformed and tested on the gel. The results were a smear on primary PCR and a semi-bright band on the correct nuceotide position (1500nt).
(9/6/13) PCR squared was preformed and tested on gel.Then gel extraction was performed on the PCR squared product since the band was at the correct position. The concentration after gel extraction was around 50ng/microliter. The 230nm. peak was high while the 260 nm. peak was low, which could account for poor cloning, but I have decided to continue to cloning next week.
Nanodrop Concentration after Gel Extraction
Gel Extraction
PCR squared
Primary and Secondary PCR
NEB 1 KB Ladder
WEEK FIVE
Primer Design (Putative acid phosphatase of H. pylori in pNIC-Bsa4)
Forward Primer:
5’ TACTTCCAATCATGGTAAAAAAGACGCTGGCATC 3’ 34 bp
GC Content 41.2%
0 mM Mg2+ Tm 62.1 oC 1.5 mM Mg2+ Tm 69.6 oC 2 mM Mg2+ Tm 70.1 oC
4 mM Mg2+ Tm 71.1oC 6 mM Mg2+ Tm 71.6 oC
Reverse Primer:
5’ AAAGCGTGGCAGAACAAAAAGTAACAGTAAAGGTGGATA 3’
Reverse complement it:
5’ TATCCACCTTTACTGTTACTTTTTGTTCTGCCACGCTT 3’ 38 bp
GC Content 39.5%
0 mM Mg2+ Tm 63.7 oC 1.5 mM Mg2+ Tm 71.5 oC 2 mM Mg2+ Tm 71.9 oC
4 mM Mg2+ Tm 72.9 oC 6 mM Mg2+ Tm 73.3 oC
WEEK FOUR
Protein Purification: (PfDXR in pNic-BSSA4)
SDS-PAGE Gel Results:
WEEK THREE
Third PCR: (pmCHERRY)
Large-Scale Protein Expression: (PfDXR in pNic-BSSA4)
pellet weight=2.8 g.
Second PCR: (pgbr22)
_
WEEK TWO
First PCR Results:
Midi-Prep DNA Sequence: (92015)
NNNNNNNNNNNNNNNTNNACTTTAGANGAGATNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATNTGGGT
ACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGAT
ATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGA
AAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGNAGTCTGCAAATCCTTTTATGATTTTCTATCAAACANAAGAGGA
AAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAATAGTAANTCNNGCGGGTTTGTTNNNGANNAA
GNNNGCAANANNAAANANGTGTNNNTNNAAAAGCGNNTTNTTTTNNGTNNNNACNNNNNCATCNCNNNNNTNCTTTNNNN
NTTGCNCCANTGNNNNNTTNNNNNNNNNANNANAAGNNNNNNNNNNNNNNNNNNNN
Midi-Prep Nanodrop:
___
WEEK ONE
DNA sequencing: (91623)
NNNNNNNNNNGNNNNNATAGAATACTCAAGCTATGCATCCNACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCG
GCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATAT
GTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAA
AGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCA
TTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAA
CTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCT
CTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTT
GCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAA
ATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACA
ACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCAC
TAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCAC
TGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTC
GCCAGCTGGCGTAATAGCGAANANGCCCGCACCGATCGCCCNTNCCCAACAGTTGCGCANCCTGAATNGNNAATGGACGC
NNCCNNNANNNNNNCATTNAGNNNGNNNNNNNNNNNNNCNCNCAGCNNGACGCNNNNNNTTNCNNCNNNNNNNNCCNNNT
NNTNNNNNTTCNNNCNNNNTNCNNNNCNCNNNNNNNNNNTNNCCNNNANNNNNNNNNGGNNNNNNNTNNGNNNCNNNNNN
NNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
Plasmid**:
Plate A: 1080 colonies / 2ng plasmid = 540 colonies per ng plasmid (pNIC BSA4)
Plate B: 456 colonies / 10ng plasmid = 45.6 colonies per ng plasmid (pNIC BSA4)
Plate C: 68 colonies / 50ng plasmid = 1.36 colonies per ng plasmid (pNIC BSA4)
Primer Dilutions: